CN114946829B - Hair follicle tissue vitrification frozen stock solution - Google Patents
Hair follicle tissue vitrification frozen stock solution Download PDFInfo
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- 210000003780 hair follicle Anatomy 0.000 title claims abstract description 124
- 238000004017 vitrification Methods 0.000 title claims abstract description 60
- 239000011550 stock solution Substances 0.000 title claims abstract description 59
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- 239000012091 fetal bovine serum Substances 0.000 claims abstract description 24
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- HDTRYLNUVZCQOY-NCFXGAEVSA-N beta,beta-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-NCFXGAEVSA-N 0.000 description 2
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- 210000002752 melanocyte Anatomy 0.000 description 2
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Abstract
The application provides a hair follicle tissue vitrification frozen stock solution which comprises the following components in percentage by volume: 18% -25% of dimethyl sulfoxide, 12% -18% of acetamide, 3% -6% of 1, 2-propylene glycol, 3% -8% of 2, 3-butanediol, 5% -7% of polyethylene glycol, 1-5% of dextran, 10% of fetal bovine serum FBS and balance of balance buffer; the hair follicle tissue vitrification frozen stock solution also comprises 0.2-0.9g/ml of trehalose; the sum of the volume percentages of all components is 100%. The hair follicle tissue vitrification frozen stock solution reduces the formation of ice crystals when the hair follicle tissue is frozen, thereby effectively preventing the hair follicle tissue cells from being frostbitten, improving the survival rate of the hair follicle tissue cells for freezing preservation, and providing corresponding nutrient substances and living environment in time when in freezing and after thawing, and improving the survival rate when in thawing, so that the survival rate of the unfrozen hair follicle tissue cells is high.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a hair follicle tissue vitrification frozen stock solution.
Background
Cryopreservation is an effective method for long-term preservation of living tissues, and mainly plays a role in protecting by reducing the metabolic rate of cells, and frostbite is the side reaction with the greatest cryoprotection, and the reasons for the frostbite are probably mechanical damage and permeability damage caused by ice crystals. At present, two main methods exist for tissue cryopreservation: quick freezing and storing, namely vitrification; and (3) a slow quick freezing storage method, namely a program cooling method. The vitrification method is to utilize vitrification cryoprotectant liquid formed by combining multiple cryoprotectants with high concentration to protect suspended cells, and directly put into liquid nitrogen for freezing and preserving. The vitrification method is widely successful in preserving animal and plant cells, but is very rare in the field of microorganism preservation; in addition, the existing vitrification frozen stock solution contains various components and is mostly composed of 5-6 compounds except water. Ethylene glycol is toxic to animals, and therefore polyethylene glycol and the like are basically used as components of vitrified frozen stock solution.
Hair follicle stem cells are one of the perifollicular sheath bulge in humans. Hair follicle stem cells belong to adult stem cells, are in a static state in vivo, and show a remarkable proliferation capacity under the in vitro culture effect. It has been found that hair follicle stem cells have a multipotent differentiation potential, which can differentiate into epidermis, hair follicle, sebaceous glands, involved in the process of wound healing in the skin. The hair follicle stem cells have high proliferation potential, are clonally grown in vitro, can be induced to differentiate into cells such as neuron cells, glial cells, smooth muscle cells, melanocytes and the like, and can differentiate into neurons, melanocytes, keratinocytes and the like when implanted into a body. Therefore, hair follicle stem cells are important for research of cell engineering, when the hair follicle stem cells are researched and developed, hair follicle tissues need to be frozen and stored, and then thawed and resuscitated when the hair follicle tissues need to be used, however, the prior art does not have vitrified frozen stock mainly used for the hair follicle tissues, and when the existing frozen stock is used for freezing the hair follicle tissues, the resuscitating effect is poor.
Disclosure of Invention
The application aims to solve the problem of providing the hair follicle tissue vitrification frozen stock solution, which can effectively prevent hair follicle tissue cells from being frostbitten when the hair follicle tissue is frozen, improve the survival rate of the hair follicle tissue cells for freezing and preserving, provide corresponding nutrient substances and survival environment in time when the hair follicle tissue cells are frozen and after the hair follicle tissue cells are thawed, improve the survival rate and ensure that the revival rate of the thawed hair follicle tissue cells is high.
In order to solve the technical problems, the application adopts the following technical scheme: the hair follicle tissue vitrification frozen stock solution comprises the following components in percentage by volume: 18% -25% of dimethyl sulfoxide, 12% -18% of acetamide, 3% -6% of 1, 2-propylene glycol, 3% -8% of 2, 3-butanediol, 5% -7% of polyethylene glycol, 1-5% of dextran, 10% of fetal bovine serum FBS and balance of balance buffer;
the hair follicle tissue vitrification frozen stock solution also comprises 0.2-0.9g/ml of trehalose;
the sum of the volume percentages of all components is 100%.
The hair follicle tissue vitrification frozen stock solution comprises the following components in percentage by volume: 18% -25% of dimethyl sulfoxide, 22% of dimethyl sulfoxide, 15% of acetamide, 5% of 1, 2-propylene glycol, 5% of 2, 3-butanediol, 5% of polyethylene glycol, 5% of dextran, 10% of fetal bovine serum FBS and balance of balance buffer solution;
the hair follicle tissue vitrification frozen stock solution also comprises 0.2-0.9g/ml of trehalose;
the sum of the volume percentages of all components is 100%.
Further, the balance buffer solution is prepared from HEPES buffer solution and basic culture medium according to the volume ratio of 1 (35-40).
Further, the basal medium is DMEM, DMEM-F12 or 1640 medium.
Further, the trehalose is one or more of alpha, alpha-trehalose, alpha, beta-trehalose and beta, beta-trehalose.
The hair follicle tissue vitrification frozen stock solution is prepared by the following steps:
1) Mixing the balance buffer solution, fetal bovine serum FBS and dextran to obtain a mixed matrix solution A;
2) Adding acetamide, 2, 3-butanediol and polyethylene glycol into the mixed matrix liquid A in the step 1) for mixing to obtain mixed matrix liquid B;
3) Adding dimethyl sulfoxide, 1, 2-propylene glycol and trehalose into the mixed matrix liquid B in the step 2) to obtain the hair follicle tissue vitrification frozen stock solution
The application of the hair follicle tissue vitrification frozen solution in the method for freezing and storing hair follicle stem cells is that the hair follicle tissue which stores the complete hair bulb part is taken and put into 5ml frozen storage pipes, 30-40 hair follicle tissues which store the complete hair bulb part are put into each frozen storage pipe, the hair follicle tissue vitrification frozen solution is added into the frozen storage pipes, and after soaking for 5min, the frozen storage pipes are transferred into liquid nitrogen for freezing and storing.
The application has the advantages and positive effects that:
1. after the hair follicle tissue vitrification frozen stock solution freezes and stores hair follicle tissue, when stem cells are extracted after thawing, the survival rate can reach more than 94 percent, and the thawed hair follicle tissue cells have high recovery rate and practical significance.
2. The vitrification frozen stock solution effectively prevents the hair follicle tissue cells from being frostbitten when freezing and storing the hair follicle tissue, improves the survival rate of the hair follicle tissue cells for freezing and storing, and provides corresponding nutrient substances and living environment in time when freezing and after thawing by matching the components, so that the survival rate of the thawed hair follicle tissue cells is high.
Drawings
FIG. 1 is a diagram showing DPC cell status in example 1 of the present application;
FIG. 2 is a graph showing DPC cell status in the blank of the present application;
FIG. 3 is a graph showing DPC cell status in comparative example 1 of the present application;
FIG. 4 is a graph showing DPC cell status in comparative example 2 of the present application;
FIG. 5 is a DSC cell state diagram of example 1 of the present application;
FIG. 6 is a diagram showing DSC cell states in a blank of the present application;
FIG. 7 is a DSC cell state diagram of comparative example 1 of the present application;
FIG. 8 is a DSC cell state diagram of comparative example 2 of the present application;
FIG. 9 is a diagram showing the status of ORSC cells in example 1 of the present application;
FIG. 10 is a graph of ORSC cell status in the blank of the application;
FIG. 11 is a graph showing the status of ORSC cells in comparative example 1 of the present application;
FIG. 12 is a graph showing the state of ORSC cells in comparative example 2 of the present application.
Detailed Description
The present application will be described in detail with reference to the following embodiments.
Example 1:
the hair follicle tissue vitrification frozen stock solution comprises the following components in percentage by volume:
18% dimethyl sulfoxide, 12% acetamide, 3% 1, 2-propanediol, 3% 2, 3-butanediol, 5% polyethylene glycol, 15% dextran, 10% fetal bovine serum FBS, 34% equilibration buffer and 0.2g/ml trehalose.
Wherein the balance buffer solution is prepared from HEPES buffer solution and DMEM according to a volume ratio of 1:35; trehalose is alpha, alpha-trehalose.
The preparation method of the hair follicle tissue vitrification frozen stock solution comprises the following steps:
1) Mixing the balance buffer solution, fetal bovine serum FBS and dextran to obtain a mixed matrix solution A;
2) Adding acetamide, 2, 3-butanediol and polyethylene glycol into the mixed matrix liquid A in the step 1) for mixing to obtain mixed matrix liquid B;
3) And adding dimethyl sulfoxide, 1, 2-propylene glycol and trehalose into the mixed matrix liquid B in the step 2) to obtain the hair follicle tissue vitrification frozen stock solution.
Example 2:
the hair follicle tissue vitrification frozen stock solution comprises the following components in percentage by volume:
25% dimethyl sulfoxide, 18% acetamide, 6% 1, 2-propanediol, 8% 2, 3-butanediol, 7% polyethylene glycol, 5% dextran, 10% fetal bovine serum FBS, 21% equilibration buffer and 0.9g/ml trehalose.
Wherein the balance buffer solution is prepared from HEPES buffer solution and DMEM according to a volume ratio of 1:40; trehalose is alpha, beta-trehalose.
The preparation procedure of the hair follicle tissue vitrification frozen stock is the same as in example 1.
Example 3:
the hair follicle tissue vitrification frozen stock solution comprises the following components in percentage by volume:
22% dimethyl sulfoxide, 15% acetamide, 5% 1, 2-propanediol, 5% 2, 3-butanediol, 5% polyethylene glycol, 5% dextran, 10% fetal bovine serum FBS, 33% equilibration buffer and 0.5g/ml trehalose.
Wherein the balance buffer solution is prepared from HEPES buffer solution and DMEM according to a volume ratio of 1:35; trehalose is beta, beta-trehalose.
The preparation procedure of the hair follicle tissue vitrification frozen stock is the same as in example 1.
Example 4:
the hair follicle tissue vitrification frozen stock solution comprises the following components in percentage by volume:
20% dimethyl sulfoxide, 12% acetamide, 5% 1, 2-propanediol, 8% 2, 3-butanediol, 7% polyethylene glycol, 3% dextran, 10% fetal bovine serum FBS, 35% equilibration buffer and 0.7g/ml trehalose.
Wherein the balance buffer solution is prepared from HEPES buffer solution and DMEM according to a volume ratio of 1:35; trehalose is alpha, beta-trehalose.
The preparation procedure of the hair follicle tissue vitrification frozen stock is the same as in example 1.
Comparative example 1:
the biological tissue cell vitrification frozen stock solution comprises the following components in percentage by volume:
21.5% dimethyl sulfoxide, 16.5% acetamide, 5.5% 1, 2-propanediol, 6.5% 2, 3-butanediol, 7% polyethylene glycol.
When the biological tissue cell vitrification frozen stock solution is prepared, the dimethyl sulfoxide, the acetamide, the 1, 2-propylene glycol, the 2, 3-butanediol and the polyethylene glycol with corresponding volumes are directly taken and uniformly mixed to obtain the biological tissue cell vitrification frozen stock solution.
Comparative example 2:
a KSR solution for use in frozen stock solution consisting of 80vol% KSR+10vol% DMSO+10vol% trehalose, wherein the concentration of KSR solution of trehalose is 2mol/L.
Experimental example:
1. freezing hair follicle tissue:
cutting hair follicle roots, cleaning, dividing 18 5ml cryopreservation pipes into six groups, respectively numbered a, b, c, d, e, f, wherein each group comprises nine hair follicle tissues for preserving complete hair bulb parts, putting 40 hair follicle tissues for preserving complete hair bulb parts into each cryopreservation pipe, and then adding 4ml hair follicle tissue vitrification cryopreservation liquid into each cryopreservation pipe; wherein three groups of the hair follicle tissue vitrification frozen stock solution prepared in the embodiment 1 is added into the frozen stock tube in the group a, the hair follicle tissue vitrification frozen stock solution prepared in the embodiment 2 is added into the frozen stock tube in the group b, the hair follicle tissue vitrification frozen stock solution prepared in the embodiment 3 is added into the frozen stock tube in the group c, the hair follicle tissue vitrification frozen stock solution prepared in the embodiment 4 is added into the frozen stock tube in the group d, the vitrification frozen stock solution prepared in the comparative example 1 is added into the frozen stock tube in the group e, and the frozen stock solution prepared in the comparative example 2 is added into the frozen stock tube in the group f; after 5min of soaking, the frozen tube was transferred to liquid nitrogen for 3 months.
2. Thawing and activating hair follicle tissue:
(1) Placing fresh culture medium in 37 deg.C water tank, heating, spraying 75% alcohol, wiping, and transferring into aseptic operation table;
(2) Taking out the frozen tube from the liquid nitrogen, putting the frozen tube into a water tank at 37 ℃ for quick thawing, shaking the frozen tube slightly to enable the frozen tube to be completely thawed within 1 minute, wiping the outer part of the tube with 75% alcohol, and transferring the tube into a sterile operation table. The thawed hair follicle tissue in a, b, c, d, e, f was divided into three groups of three branches for extraction of DPC stem cells, DSC stem cells and ORSC stem cells, respectively.
3. Preparation of cultured DPC stem cells:
1. blank control group: 120 hair follicle tissues storing the whole hair bulb are divided into three groups g1, g2 and g3 respectively, and the three groups are used as blank control groups for preparing and culturing DPC stem cells.
2. Culturing:
the hair follicle tissue with intact hair bulb was washed 2-3 times with a double antibody-containing PBS solution.
The entire hair papilla was separated under a microscope with a surgical knife head, the remaining hair matrix was removed, and the tissue was transferred to a new PBS-containing petri dish with a pipette. Adding 0.1% collagenase IV, digesting for 1-2 h at 37deg.C for 10min, blowing the tissue with a suction tube, picking out single hair papilla with a pipette after the dissociation of dermal sheath tissue around hair papilla tissue is observed, placing into PBS solution containing double antibodies, cleaning, placing into culture dish containing hair follicle DPC medium after 30 s, and placing into 37 deg.C and 5% incubator for culturing.
DPC was seen to climb out of the papilla for about 10 days, followed by cell exchange every 3 days. After 15 days of culture, when the cells are paved at about 80-90% of the area of the bottom of the culture dish, carrying out pancreatin digestion and passage; subculture for 2 days.
The state diagram of the thawed primary DPC cells is shown in fig. 1 to 4, wherein fig. 1 is the state diagram of DPC cells in example 1, fig. 2 is the state diagram of DPC cells in a blank group, fig. 3 is the state diagram of DPC cells in comparative example 1, and fig. 4 is the state diagram of DPC cells in comparative example 2.
3. Cell count and activity detection of amplified DPC stem cells
Principle of:
the cell count is carried out by using a blood cell count plate, which generally has two chambers, 9 of which are each carved with 1mm 2 The large square, in which the square at 4 corners is further carved with 16 cells, each 0.1mm deep. When the cover glass is covered on the chamber, the volume of each large square is 1mm 2 ×0.1mm＝1.0*10 -4 ml. In use, the number of cells within each large square is counted, multiplied by the dilution factor, and multiplied by 10 4 I.e.the number of cells per ml. The number of cells in square squares was calculated in this experiment.
Cell viability detection: the dye can permeate into dead cells to generate color, while living cells cannot permeate into the cells to generate color due to complete cell membranes. The experiment used a trypan blue dye.
Cell viability = viable cell number/(viable cell number + dead cell number) ×100%.
The method comprises the following specific steps:
(1) Mu.l of the cell suspension was mixed with an equal volume of 50. Mu.l of trypan blue and homogenized in a 1.5ml microcentrifuge tube.
(2) A small amount of the mixture (about 15. Mu.l) was added from the well above the chamber of the blood cell counting tray, covered with a cover slip, and the living cells were not stained and the dead cells were blue when observed under a 100-fold inverted microscope.
(3) Counting the living cells and dead cells in the step (2), and then calculating the cell survival rate.
Note that: when the counting plate counts, the optimal concentration is 5-10 x 10 5 If the concentration is higher, the cells/ml may be removed and the fraction may be diluted or counted after serial dilution.
The cell numbers and cell viability of each group are shown in table 1:
TABLE 1DPC Stem cell viability calculation data
From the experimental data in table 1, it can be seen that:
comparing the experimental data of the examples 1-4 of the application with the experimental data of the blank control group, the cell survival rate of the hair follicle tissue frozen by the hair follicle tissue vitrification frozen solution of the application after thawing is higher than 94%, which is close to the cell survival rate of the fresh hair follicle tissue.
As can be seen from comparison of experimental data of the embodiment of the application with experimental data of the comparative example 1 and experimental data of the comparative example 2, the performance of the hair follicle tissue vitrification frozen stock solution of the application is far higher than that of the frozen stock solution in the prior art, and the survival rate of the hair follicle tissue after thawing is high and is at least improved by more than 10% compared with that of cells in the prior art.
From the experimental data in table 1, it can be seen that the survival rate of DPC stem cells prepared from the hair follicle tissue frozen by using the frozen stock solution is close to that of fresh hair follicle tissue after resuscitation, and thus, the hair follicle tissue vitrification frozen stock solution claimed by the application has good frozen stock effect and is suitable for hair follicle tissue.
4. Preparation of cultured DSC stem cells:
1. blank control group: 120 hair follicle tissues storing the whole hair bulb are divided into three groups of g4, g5 and g6, and the three groups are used as blank control groups for preparing and culturing DPC stem cells.
2. Culturing:
the hair follicle tissue with intact hair bulb was washed 2-3 times with a double antibody-containing PBS solution.
The entire hair papilla was separated under a microscope with a surgical knife head, the remaining hair matrix was removed, and the tissue was transferred to a new PBS-containing petri dish with a pipette. Adding 0.1% collagenase IV, digesting for 1h-2h at 37 ℃ for 10min at intervals, blowing the tissue with a suction tube, after the dermal sheath tissue around the papilla tissue is completely dissociated, selecting single papilla with a pipette under a microscope, centrifuging the residual digestive juice, collecting DSC cells, washing with PBS solution for 2 times, placing into a culture dish containing hair follicle DSC medium, and placing into a 5% incubator at 37 ℃ for culturing.
DSC adherent growth was seen around 2 days, followed by cell exchange every 3 days. After 15 days of culture, when the cells are paved at about 80-90% of the area of the bottom of the culture dish, carrying out pancreatin digestion and passage; subculture for 2 days.
The state diagram of the primary DSC cells after thawing is shown in FIGS. 5 to 8, wherein FIG. 5 is the DSC cell state diagram in example 1, FIG. 6 is the DSC cell state diagram in the blank group, FIG. 7 is the DSC cell state diagram in comparative example 1, and FIG. 8 is the DSC cell state diagram in comparative example 2.
3. Cell count and activity detection of amplified DSC stem cells
Principle of:
the cell count is carried out by using a blood cell count plate, which generally has two chambers, 9 of which are each carved with 1mm 2 The large square, in which the square at 4 corners is further carved with 16 cells, each 0.1mm deep. When the cover glass is covered on the chamber, the volume of each large square is 1mm 2 ×0.1mm＝1.0*10 -4 ml. In use, the number of cells within each large square is counted, multiplied by the dilution factor, and multiplied by 10 4 I.e.the number of cells per ml. The number of cells in square squares was calculated in this experiment.
Cell viability detection: the dye can permeate into dead cells to generate color, while living cells cannot permeate into the cells to generate color due to complete cell membranes. The experiment used a trypan blue dye.
Cell viability = viable cell number/(viable cell number + dead cell number) ×100%.
The method comprises the following specific steps:
(4) Mu.l of the cell suspension was mixed with an equal volume of 50. Mu.l of trypan blue and homogenized in a 1.5ml microcentrifuge tube.
(5) A small amount of the mixture (about 15. Mu.l) was added from the well above the chamber of the blood cell counting tray, covered with a cover slip, and the living cells were not stained and the dead cells were blue when observed under a 100-fold inverted microscope.
(6) Counting the living cells and dead cells in the step (2), and then calculating the cell survival rate.
Note that: when the counting plate counts, the optimal concentration is 5-10 x 10 5 If the concentration is higher, the cells/ml may be removed and the fraction may be diluted or counted after serial dilution.
The cell numbers and cell viability of each group are shown in table 2:
TABLE 2DSC Stem cell survival calculation data
From the experimental data in table 2, it can be seen that:
comparing the experimental data of the examples 1-4 with the experimental data of the blank control group, the cell survival rate of the hair follicle tissue frozen by the hair follicle tissue vitrification frozen solution is higher after thawing, and the cell survival rate of the hair follicle tissue frozen by the hair follicle tissue vitrification frozen solution can reach 94% or more and is close to that of the fresh hair follicle tissue.
As can be seen from comparison of experimental data of the embodiment of the application with experimental data of the comparative example 1 and experimental data of the comparative example 2, the performance of the hair follicle tissue vitrification frozen stock solution of the application is far higher than that of the frozen stock solution in the prior art, and the survival rate of the hair follicle tissue after thawing is high and is at least improved by more than 10% compared with that of cells in the prior art.
From the experimental data in table 2, it can be seen that the survival rate of DSC stem cells prepared from the hair follicle tissue frozen by using the frozen stock solution is close to that of fresh hair follicle tissue after resuscitation, so that the hair follicle tissue vitrification frozen stock solution disclosed by the application has good frozen stock effect and is suitable for hair follicle tissue.
5. Preparation of cultured ORSC Stem cells
1. Blank control group: 120 hair follicle tissues for storing the whole hair bulb part are divided into three groups g7, g8 and g9 respectively and used as blank control groups for preparing and culturing DPC stem cells
2. Culturing:
the tissue was washed 2-3 times with PBS containing the diabody.
Digestion with 0.25% Dispase enzyme overnight at 4deg.C, half an hour ahead of the next day, and incubation in 37℃incubator.
Hair follicle tissue was washed with ophthalmic forceps in a petri dish containing a double antibody in PBS. Mao Ganshun hair follicles were clamped out, residual hair matrix was removed, placed in 2ml pancreatin containing 0.5% EDTA, digested at 37 ℃ for 5-10min, and 2ml fbs was added to terminate the digestion. The supernatant was centrifuged and the pellet was resuspended in 4ml of ORSC medium and added to a petri dish containing follicular ORSC medium and incubated in a 5% incubator at 37 ℃.
ORSC wall-attached growth was seen around 2 days, followed by cell exchange every 3 days. After 15 days of culture, when the cells are spread to be full of about 80-90% of the area of the bottom of the culture dish, pancreatin digestion passage is carried out. Subculture for 2 days.
The state diagram of the primary ORSC cells after thawing is shown in FIGS. 9-12, wherein FIG. 9 is the state diagram of the ORSC cells in example 1, FIG. 10 is the state diagram of the ORSC cells in the blank, FIG. 11 is the state diagram of the ORSC cells in comparative example 1, and FIG. 12 is the state diagram of the ORSC cells in comparative example 2.
3. Cell count and viability detection of amplified ORSC stem cells
Principle of:
the cell count is carried out by using a blood cell count plate, which generally has two chambers, 9 of which are each carved with 1mm 2 The large square, in which the square at 4 corners is further carved with 16 cells, each 0.1mm deep. When the cover glass is covered on the chamber, the volume of each large square is 1mm 2 ×0.1mm＝1.0*10 -4 ml. In use, the number of cells within each large square is counted, multiplied by the dilution factor, and multiplied by 10 4 I.e. eachCell number in ml. The number of cells in square squares was calculated in this experiment.
Cell viability detection: the dye can permeate into dead cells to generate color, while living cells cannot permeate into the cells to generate color due to complete cell membranes. The experiment used a trypan blue dye.
Cell viability = viable cell number/(viable cell number + dead cell number) ×100%.
The method comprises the following specific steps:
(7) Mu.l of the cell suspension was mixed with an equal volume of 50. Mu.l of trypan blue and homogenized in a 1.5ml microcentrifuge tube.
(8) A small amount of the mixture (about 15. Mu.l) was added from the well above the chamber of the blood cell counting tray, covered with a cover slip, and the living cells were not stained and the dead cells were blue when observed under a 100-fold inverted microscope.
(9) Counting the living cells and dead cells in the step (2), and then calculating the cell survival rate.
Note that: when the counting plate counts, the optimal concentration is 5-10 x 10 5 If the concentration is higher, the cells/ml may be removed and the fraction may be diluted or counted after serial dilution.
The cell numbers and cell viability of each group are shown in table 1:
TABLE 3ORSC Stem cell viability calculation data
From the experimental data in table 3, it can be seen that:
comparing the experimental data of the examples 1-4 of the application with that of the blank control group, the cell survival rate of the hair follicle tissue frozen by the hair follicle tissue vitrification frozen solution of the application after thawing is higher, the cell survival rate of the hair follicle tissue is up to 96% and above, and is close to that of the fresh hair follicle tissue.
As can be seen from comparison of experimental data of the embodiment of the application with experimental data of the comparative example 1 and experimental data of the comparative example 2, the performance of the hair follicle tissue vitrification frozen stock solution of the application is far higher than that of the frozen stock solution in the prior art, and the survival rate of the hair follicle tissue after thawing is high and is at least improved by more than 10% compared with that of cells in the prior art.
From the experimental data in table 3, it can be seen that the survival rate of the ORSC stem cells prepared from the hair follicle tissue frozen by using the frozen stock solution is close to that of fresh hair follicle tissue after resuscitation, so that the hair follicle tissue vitrification frozen stock solution disclosed by the application has a good frozen stock effect and is suitable for hair follicle tissue.
The comparison of the several groups of experiments proves that the vitrified frozen stock solution has better application effect on hair follicle tissues, and the survival rate of the hair follicle tissues frozen by using the vitrified frozen stock solution can reach more than 94% when stem cells are extracted after thawing, so that the vitrified frozen stock solution has practical significance; therefore, the vitrified frozen stock solution can effectively prevent the hair follicle tissue cells from being frostbitten, improves the survival rate of the hair follicle tissue cells for freezing preservation, and has high recovery rate of the hair follicle tissue cells after thawing.
The foregoing describes the embodiments of the present application in detail, but the description is merely a preferred embodiment of the present application and should not be construed as limiting the scope of the application. All equivalent changes and modifications within the scope of the present application are intended to be covered by the present application.
Claims (6)
1. A hair follicle tissue vitrification frozen stock solution is characterized in that: comprises the following components in percentage by volume: 18% -25% of dimethyl sulfoxide, 12% -18% of acetamide, 3% -6% of 1, 2-propylene glycol, 3% -8% of 2, 3-butanediol, 5% -7% of polyethylene glycol, 1-5% of dextran, 10% of fetal bovine serum FBS and balance of balance buffer;
the hair follicle tissue vitrification frozen stock solution also comprises 0.2-0.9g/ml of trehalose;
the sum of the volume percentages of all components is 100%;
the hair follicle tissue vitrification frozen stock solution is prepared by the following steps:
1) Mixing the balance buffer solution, fetal bovine serum FBS and dextran to obtain a mixed matrix solution A;
2) Adding acetamide, 2, 3-butanediol and polyethylene glycol into the mixed matrix liquid A in the step 1) for mixing to obtain mixed matrix liquid B;
3) And adding dimethyl sulfoxide, 1, 2-propylene glycol and trehalose into the mixed matrix liquid B in the step 2) to obtain the hair follicle tissue vitrification frozen stock solution.
2. The hair follicle tissue vitrification frozen stock solution as recited in claim 1, wherein: comprises the following components in percentage by volume: 22% of dimethyl sulfoxide, 15% of acetamide, 5% of 1, 2-propylene glycol, 5% of 2, 3-butanediol, 5% of polyethylene glycol, 5% of dextran, 10% of fetal bovine serum FBS and balance of balance buffer;
the hair follicle tissue vitrification frozen stock solution also comprises 0.2-0.9g/ml of trehalose;
the sum of the volume percentages of all components is 100%.
3. A hair follicle tissue vitrification frozen stock solution according to claim 1 or 2, characterized in that: the equilibrium buffer solution is prepared from HEPES buffer solution and basic culture medium according to the volume ratio of 1 (35-40).
4. A hair follicle tissue vitrification frozen stock solution as in claim 3, wherein: the basal medium is DMEM, DMEM-F12 or 1640 medium.
5. A hair follicle tissue vitrification frozen stock solution according to claim 1 or 2, characterized in that: the trehalose is one or more of alpha, alpha-type trehalose, alpha, beta-type trehalose and beta, beta-type trehalose.
6. Use of the hair follicle tissue glass frozen stock solution according to any one of claims 1-5 in a method of freezing hair follicle stem cells, characterized in that: placing the hair follicle tissues for storing the complete hair bulb into 5ml freezing pipes, placing 30-40 hair follicle tissues for storing the complete hair bulb into each freezing pipe, adding the hair follicle tissue vitrification freezing solution into the freezing pipes, soaking for 5min, and transferring the freezing pipes into liquid nitrogen for freezing.
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Denomination of invention: A type of hair follicle tissue vitrification cryopreservation solution Granted publication date: 20231103 Pledgee: Bank of Jiangsu Limited by Share Ltd. Hangzhou branch Pledgor: ZHEJIANG HEALTHFUTURE BIOMEDICAL TECHNOLOGY CO.,LTD. Registration number: Y2024980002379 |
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